Biomedical subjects

Khampoune Sayasith

Publications and source records attributed to Khampoune Sayasith.

8 recordsLinked to original sources

PubMed2006

Molecular cloning and gonadotropin-dependent regulation of equine prostaglandin F2alpha receptor in ovarian follicles during the ovulatory process in vivo.

The progressive rise in gonadotropins prior to ovulation triggers a marked increase in intrafollicular levels of prostaglandin F(2alpha)(PGF(2alpha)), which is known to interact with PGF(2alpha) receptor (FP). Little is known about the regulation of FP during ovulation. This study was undertaken to characterize the equine FP and its gonadotropin-dependent regulation in preovulatory follicles prior to ovulation. The full-length equine FP encodes a 366-amino acid protein that is 82-93% homologous to other species. Using semi-quantitative RT-PCR/Southern blot, we showed that FP mRNA expression was low in follicles obtained before hCG treatment (0h) and at 24, but increased at 12 and 36h post-hCG (P<0.05). This expression was regulated in both follicular cells, with high levels of the transcript at 33 and 36h post-hCG in granulosa cells, and at 12, 30 and 33h post-hCG in theca cells (P<0.05). Immunohistochemistry confirmed the induction of FP protein in both follicular cells after hCG, and immunoblotting revealed the increase of FP protein in preovulatory follicles 36h post-hCG. High levels of FP mRNA were detected in the corpora lutea and heart, but very low or undetectable in other tissues. This study reports for the first time the expression of FP and its up-regulation by hCG in preovulatory follicles prior to ovulation. FP regulation was occurred in different pattern than that observed in other species, suggesting a distinct and species-specific follicular control of FP expression during ovulation, and a potential involvement of PGF(2alpha), acting on granulosa and theca cells, in the ovulatory process.

Amino Acid Sequence↗

PubMed2006

Expression of phospholipase A2 group IVA (PLA2G4A) is upregulated by human chorionic gonadotropin in bovine granulosa cells of ovulatory follicles.

Prostaglandins are required for the ovulatory process, and their biosynthesis depends on the initial release of arachidonic acid from membrane phospholipids. We hypothesized that phospholipase A2 group IVA (PLA2G4A) expression is upregulated in granulosa cells (GC) at ovulation. We have characterized bovine PLA2G4A cDNA, and investigated its spatiotemporal regulation at the mRNA and protein levels in hCG-induced ovulatory follicles and in vitro, using forskolin-stimulated GC. Regulation of PLA2G4A mRNA expression was studied in GC obtained from bovine follicles collected at different developmental stages: small follicles (2-4 mm), dominant follicles at Day 5 (D5) of the estrous cycle, ovulatory follicles 24 h following injection of hCG, and corpus luteum at D5. PLA2G4A mRNA increased by 14-fold in GC of hCG-stimulated versus dominant follicles (P < 0.0001). Follicular walls obtained from ovulatory follicles recovered at 0, 6, 12, 18, and 24 h post-hCG injection showed an initial 16-fold increase in PLA2G4A transcript at 12 h that reached a 45-fold increase at 24 h, as compared to 0 h (P < 0.0001). Immunoblots of GC extracts showed an initial induction of the PLA2G4A protein at 18 h post-hCG, reaching a maximum at 24 h. Immunohistochemistry observations showed that PLA2G4A signal was mainly observed in mural GC compared to antral GC in hCG-stimulated follicles. Stimulation of cultured bovine GC with 10 microM of forskolin caused an increase in PLA2G4A mRNA and protein. Ovulation is associated with an LH/hCG-dependent induction of PLA2G4A in GC via the adenylyl cyclase/cAMP pathway.

Adenylyl Cyclases↗

PubMed2006

Characterization of bovine early growth response factor-1 and its gonadotropin-dependent regulation in ovarian follicles prior to ovulation.

Early growth response factor-1 (EGR-1) is a transcription factor that is involved in the transactivation of several genes. The objective of this study was to characterize gonadotropin-dependent regulation of bovine EGR-1 in preovulatory follicles prior to ovulation. Bovine EGR-1 cDNA was obtained by RT-PCR, 5'- and 3'-RACE, its open reading frame composed of 1623 bp, and its coding region encodes a 540-amino acid protein that displays 62-93% identity to known mammalian homologs. The regulation of EGR-1 mRNA was studied in bovine preovulatory follicles which were isolated 0-24 h post-hCG using semiquantitative RT-PCR/Southern blot. Results revealed that the levels of EGR-1 mRNA were very low in follicles at 0 h, markedly increased at 6 h (P < 0.05) when compared to 0 h, and decreased between 12 and 24 h post-hCG. High levels of the EGR-1 mRNA were also observed in corpus luteum, uterus, kidney, pituitary, and spleen, moderate and low in other bovine tissues tested. Analyses performed on isolated preparations of granulosa and theca cells indicated that EGR-1 mRNA was regulated in both cell types, with a predominant expression in granulosa cells. Immunohistochemistry on sections of preovulatory follicles isolated before and after hCG confirmed its protein expression in granulosa cells, 24 h post-hCG. Studies of EGR-1 regulation in primary granulosa cells cultured with forskolin showed that levels of EGR-1 mRNA were low at 0 h, highly increased at 6 h post-forskolin (P < 0.05), and declined to steady state thereafter. Immunoblotting confirmed forskolin-induced EGR-1 protein in cultures. Interestingly, overexpression of EGR-1 increased the levels of mRNA for prostaglandin (PG) G/H synthase-2 (PGHS-2), PG E synthase (PGES), PG E2 receptor (EP2), LH receptor (LH-R), but not for cytochrome P450-side chain cleavage (P450scc), and cytochrome P450 aromatase (P450arom) in granulosa cultures. Thus, this study reports for the first time, a gonadotropin-dependent induction of follicular EGR-1 prior to ovulation in large monoovulatory species and its stimulating effect on the expression of genes known to be involved in prostaglandin biosynthesis pathway, thereby suggesting its potential involvement in the regulation of preovulatory events in cattle.

Amino Acid Sequence↗

PubMed2005

Role of upstream stimulatory factor phosphorylation in the regulation of the prostaglandin G/H synthase-2 promoter in granulosa cells.

To investigate the role of USF phosphorylation in the regulation of the PGHS-2 promoter in granulosa cells, promoter activity assays were performed in primary cultures of bovine granulosa cells transfected with the chimeric PGHS-2 promoter/luciferase (LUC) construct -149/-2PGHS-2.LUC. Transfections were done in the absence or presence of forskolin; the protein kinase A (PKA) inhibitor H-89; or an expression vector encoding USF1, USF2, the catalytic subunit of PKA (cPKA), or a PKA inhibitor protein (PKI). Electrophoretic mobility shift assays were performed to study USF/DNA interactions using granulosa cell nuclear extracts and a 32P-labeled proximal PGHS-2 promoter fragment containing the E-box element. The results show that forskolin stimulation and cPKA overexpression caused a marked and significant increase in USF-dependent DNA binding and PGHS-2 promoter activities (p < 0.05). In contrast, both activities were decreased by H-89 treatment or PKI overexpression. Reverse transcription-PCR analyses revealed that these treatments had similar effects on endogenous PGHS-2 mRNA levels in granulosa cells. Cotransfection studies with a USF2 mutant lacking N-terminal activation domains (U2Delta1-220) repressed forskolin-, cPKA-, and USF-dependent PGHS-2 promoter activities. Electrophoretic mobility shift assays showed that U2Delta1-220 was able to compete with full-length USF proteins and to saturate the E-box element. Immunoprecipitation/Western blot analyses revealed an increase in the levels of phosphorylated USF1 and USF2 after forskolin treatment, whereas chromatin immunoprecipitation assays showed that binding of USF proteins to the endogenous PGHS-2 promoter was stimulated by forskolin. Site-directed mutagenesis of a consensus PKA phosphorylation site within USF proteins abolished their transactivating capacity. Collectively, these results characterize the role of USF phosphorylation in PGHS-2 expression and identify the phosphorylation-dependent increase in USF binding to the E-box as a putative molecular basis for the increase in PGHS-2 promoter transactivation in granulosa cells.

Animals↗

PubMed2004

Molecular characterization of equine P-selectin (CD62P) and its regulation in ovarian follicles during the ovulatory process.

Ovulation is accompanied by a marked infiltration of leukocytes into thecal layers after the gonadotropin surge. P-selectin is known to play a critical role in the initial steps of leukocyte recruitment from the bloodstream during inflammation. Thus, the objective was to investigate the potential regulation of P-selectin by gonadotropins in equine preovulatory follicles. The full-length equine P-selectin cDNA was cloned by a combination of reverse transcription-polymerase chain reaction (RT-PCR) and 5'- and 3'-rapid amplification of cDNA ends. Results showed that equine P-selectin cDNA encodes an 829-amino acid protein that is highly conserved when compared to the human protein (80% identity). Semiquantitative RT-PCR/Southern blot analyses were performed to study the regulation of P-selectin transcript in preovulatory follicles isolated during estrus at 0, 12, 24, 30, 33, 36, and 39 h after an ovulatory dose of hCG (ovulation occurs between 39 and 42 h post-hCG in this model). Results showed that levels of P-selectin mRNA remained very low or undetectable throughout the ovulatory process in extracts prepared from the granulosa cell layer. In contrast, a significant increase in P-selectin transcript was observed between 30 and 39 h post-hCG in extracts obtained from thecal layers (P < 0.05). Likewise, immunohistochemistry revealed an increase of immunoreactive P-selectin protein in the vascular endothelium present in thecal layers of follicles isolated 36 and 39 h post-hCG. Thus, the present study describes, to our knowledge for the first time, the primary structure of equine P-selectin and the regulation of P-selectin transcript and protein in follicular thecal endothelial cells before ovulation.

Amino Acid Sequence↗

PubMed2004

Cyclooxygenase-2 and its role in ovulation: a 2004 account.

The pre-ovulatory surge of gonadotrophins triggers a marked and obligatory increase in follicular prostaglandin synthesis prior to ovulation, and the cyclooxygenase (COX) enzyme is a key rate-limiting step in the biosynthesis of prostaglandins. In the early 1990s, the pre-ovulatory rise in follicular prostaglandin synthesis was shown to result from the selective induction of a novel COX isoform, now referred to as COX-2. Differences in the time-course of COX-2 induction in species with a short versus a long ovulatory process suggest that the enzyme could be a molecular determinant that sets the alarm of the mammalian ovulatory clock. Some of the fine molecular mechanisms involved in the transcriptional activation of the COX-2 gene in granulosa cells have also been elucidated. The binding of trans-activating upstream stimulatory factors (USF) to a consensus E-box cis-element in the proximal region of the promoter was shown to play a predominant role in COX-2 transcription. Studies showed that COX-2 expression could also serve as a valuable marker for follicular commitment to ovulation during hyperstimulatory cycles. This paper presents a comprehensive review of the events that led to the characterization of COX-2 in pre-ovulatory follicles, updates current concepts on the control of COX-2 expression in pre-ovulatory follicles, and addresses the consequences of COX-2 inhibition to women fertility and potential implications of COX-2 expression in ovarian cancer.

Amino Acid Sequence↗

PubMed2003

Molecular characterization and role of bovine upstream stimulatory factor 1 and 2 in the regulation of the prostaglandin G/H synthase-2 promoter in granulosa cells.

The transcriptional activation of the prostaglandin G/H synthase-2 (PGHS-2) gene in granulosa cells is required for ovulation. To directly study the ability of upstream stimulatory factor 1 (USF1) and USF2 to trans-activate the bovine PGHS-2 promoter in granulosa cells, USF1 or USF2 expression vectors were cotransfected with the PGHS-2/luciferase (LUC) chimeric construct, -149/-2PGHS-2.LUC. Results revealed that overexpression of USF1 or USF2 caused a marked and significant increase in basal and forskolin-inducible promoter activities (p<0.05), and these effects were dependent on the presence of a consensus E-box cis-element within the promoter fragment. Co-transfections with different N- and C-terminal truncated USF mutants led to significant reductions in promoter activation, as compared with full-length constructs (p<0.05), thus allowing identification of putative bovine USF functional domains. Overexpression of a USF2 truncated mutant lacking the first 220 residues (U2Delta1-220) acted as a dominant negative mutant and blocked endogenous and USF-stimulated PGHS-2 promoter activation. Interestingly, transfections with U2Delta1-220 blocked the forskolin-dependent induction of PGHS-2 mRNA in granulosa cells, whereas transfections with full-length USF2 increased PGHS-2 transcript levels. Immunoblot analyses confirmed overexpression of full-length and truncated USF proteins, and electrophoretic mobility shift assays (EMSAs) and supershift EMSAs established that the observed effects were dependent on specific interactions between USF proteins and the consensus E-box cis-element. Stimulation of cells with forskolin increased, whereas treatment of extracts with phosphatase decreased USF binding activities to the E-box. Thus, this study presents for the first time direct evidence for a role of USF proteins in the regulation of the PGHS-2 promoter in preovulatory granulosa cells.

Amino Acid Sequence↗

PubMed2001

Targeting HIV-1 integrase.

Human immunodeficiency virus Type 1 (HIV-1) integrase is an essential enzyme for the obligatory integration of the viral DNA into the infected cell chromosome. As no cellular homologue of HIV integrase has been identified, this unique HIV-1 enzyme is an attractive target for the development of new therapeutics. Treatment of HIV-1 infection and AIDS currently consists of the use of combinations of HIV-1 inhibitors directed against reverse transcriptase (RT) and protease. However, their numerous side effects and the rapid emergence of drug-resistant variants limit greatly their use in many AIDS patients. In principle, inhibitors of the HIV-1 integrase should be relatively non-toxic and provide additional benefits for AIDS chemotherapy. There have been many major advances in our understanding of the molecular mechanism of the integration reaction, although some critical aspects remain obscure. Several classes of compounds have been screened and further scrutinised for their inhibitory properties against the HIV integrase; however, there are currently no useful inhibitors available clinically for the treatment of AIDS patients. This review describes the current knowledge of the biological functions of the HIV-1 integrase and reports the major classes of integrase inhibitors identified to date.

Journal Article↗